The infection of mammalian cells with herpes simplex virus type 1 (HSV-1) results in dramatic alterations to the host cell nucleus, so that viral genes are expressed at high levels while cellular genes are nearly completely suppressed. These changes are mediated by a small set of viral regulatory proteins that are amenable to biochemical and genetic analysis. This proposal focuses on ICP27, one of the key HSV-1 regulators. We will focus on understanding how ICP27 post-transcriptionally activates viral gene expression during infection. In Specific Aim 1, we will use the viral glycoprotein C (gC) gene as an experimental model to study how ICP27 induces responsive genes. A transfection assay will be used to map ICP27-response elements in the gC gene. We will also test whether ICP27 binds to sequences in gC mRNA. Potential ICP27 response elements or RNA-binding sites in the gC gene will be mutated in the context of recombinant viruses to ask whether they function during viral infection. In Specific Aim 2, we will characterize the nuclear export of ICP27, which is likely crucial for its ability to activate viral genes. We hypothesize that ICP27 interacts with and utilizes two distinct cellular export systems: CRM1 and REF/TAP. We will use a genetic approach to test this, by engineering and characterizing viral ICP27 mutants that have lost the ability to interact with CRM1 and/or REF. We will also study how certain ICP27 mutations confer viral resistance to leptomycin B, a CRM1inhibitor. In Specific Aim 3, we will identify other HSV-1 genes that modulate ICP27 function and expression. To do this, we will characterize two second-site suppressor mutants that we have isolated, M16R and dLeuR. Both viruses appear to have extragenic mutations that suppress ICP27defects. By identifying the genes involved, we will define viral factors which regulate ICP27 function, or compensate for its loss. Overall, this research will increase our understanding of pathogenic herpesviruses and provide novel insight into post-transcriptional gene regulation.